Our long-term goal is to determine the role of prostaglandin synthesis in cellular growth control. Several recent studies indicate the importance of prostaglandin endoperoxide synthase, type 2 (PGHS-2) in cellular homeostasis. Deletion of the PGHS-2 gene results in severe renal pathology in mice. In contrast, overexpression of PGHS-2 results in resistance to apoptosis which may contribute to carcinogenesis. These observations suggest potential mechanisms to explain the observation that inhibition of prostaglandin synthesis decreases cancer risk in humans. To approach this long-term goal we will determine the role of lipid mediators in the regulation of prostaglandin synthesis. In our previous studies, we have used the Madin-Darby canine kidney cell line to study prostaglandin synthesis in response to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We find that TPA stimulates phosphatidic acid (PA) production by phospholipase D (PLD). In TPA-stimulated cells, the PA produced by PLD is required for membrane association of Raf-1 and for induction of PGHS-2 gene expression. In contrast, induction of PGHS-2 by epidermal growth factor is not dependent on PLD or protein kinase C (PKC). These data indicate that there are distinct pathways that result in PGHS-2 induction. One of the pathways apparently requires PKC, PLD and Raf-1. Another pathway requires neither PKC nor PLD but may require Raf-1 or a similar kinase activity. To further define these pathways for the control of PGHS-2 synthesis we will pursue the following specific aims: 1) to determine the role of phospholipase D in protein kinase C-dependent signalling and 2) to define the role of Raf-1 in the control of prostaglandin synthesis. Together these studies will give us a better understanding of the control of prostaglandin synthesis and lead to further studies of the role of PGHS-2 in growth control. In addition, new targets for therapeutic intervention should become apparent that can be used to develop treatment strategies that are more selective and have less adverse effects than those currently available.